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Additional file 12: Fig. S7. Characterisation of the recombinant bacterial CAZymes encoded by the endosymbionts. DNS reducing sugars assays showing activities on a number of substrates for LpsGH5_8, LpsGH11, LpsGH134a and LpsGH134b. CMC = carboxymethyl cellulose, LBG = locust bean gum. The nanomoles of sugars released by the different proteins cannot be compared quantitatively as different amounts were used for the assay. File format .DOCX.

Additional file 8: Table S2. CAZy families. List of the CAZy families mentioned in the results, with the description of the activities that have been recorded for enzymes listed in each family. The information has been gathered from the CAZy database ( http://www.cazy.org ). File format .DOCX.

Additional file 2: Fig. S2. Searching for the opening of the duct of Deshayes in the shipworm digestive system. Left, transverse sections through the shell valve, from the anterior to posterior adductor muscle (A-E). Right, 3D rendered model of the whole shipworm, with the green highlighted region showing the position of the transverse cross section displayed on the left. File format .DOCX.

Additional file 6: Fig. S6. Immunogold labelling of LpsGH5_8 in L. pedicellatus using pre-immune serum (negative control). A) SEM image illustrating the anatomical position of the gills, food groove, caecum and mouth in the shipworm body. B-D) Immunogold labelling of the lumen of the food groove (B), lumen of the caecum (C), and the gills (D) performed with pre-immune serum. No gold particles are observed in any of the samples. B = bacteria. File format .DOCX.

Additional file 7: Table S1. Details of the proteins identified through proteomic analysis of the crystalline style. The annotation was performed against the NCBI non-redundant database, the signal peptides were identified with the Signal P4.1 server and the CAZy domains were searched using dbCAN. Many of the contigs were out of frame or not full length (particularly the bacterial ones) and therefore the identification of the signal peptide was not possible. File format .DOCX.

Additional file 4: Fig. S4. Bacterial symbionts in L. pedicellatus. A) TEM image of a gill bacteriocyte. Arrows indicate the numerous rod-shaped bacteria in the cell. B) Close-up view of gill bacteria (TEM). C) Detailed TEM showing a cross section of a gill bacterium and some of its features (periplasmic space and membranes). File format .DOCX.

Additional file 5: Fig. S5. SDS-PAGE and nitrocellulose western blot obtained by loading 0.6 ��g of the recombinant purified bacterial LpsGH5_8 (without the appended CBM). A) Coomassie Brilliant Blue stained SDS-PAGE gel of the purified protein. B) Western blot detection performed with the purified pre-immune serum. C) Western blot detection performed with the purified anti-protein serum. File format .DOCX.

Additional file 3: Fig. S3. Scanning electron microscopy of L. pedicellatus. A dissected specimen with the mantle removed, showing the food groove connecting the gills to the mouth. File format .DOCX.

Additional file 9: Table S3. CAZy families of the caecum. Table showing the relative abundance (calculated from the emPAI score) of eukaryotic and prokaryotic CAZy families identified in the proteomic analysis of the caecum content of L. pedicellatus. File format .DOCX.

Woody (lignocellulosic) plant biomass is an abundant renewable feedstock, rich in polysaccharides that are bound into an insoluble fiber composite with lignin. Marine crustacean woodborers of the genus Limnoria are among the few animals that can survive on a diet of this recalcitrant material without relying on gut resident microbiota. Analysis of fecal pellets revealed that Limnoria targets hexose-containing polysaccharides (mainly cellulose, and also glucomannans), corresponding with the abundance of cellulases in their digestive system, but xylans and lignin are largely unconsumed. We show that the limnoriid respiratory protein, hemocyanin, is abundant in the hindgut where wood is digested, that incubation of wood with hemocyanin markedly enhances its digestibility by cellulases, and that it modifies lignin. We propose that this activity of hemocyanins is instrumental to the ability of Limnoria to feed on wood in the absence of gut symbionts. These findings may hold potential for innovations in lignocellulose biorefining., Marine woodborers can digest woody biomass without the help of gut microbiota but the mechanism has remained unclear. Here, the authors provide evidence that the woodborer’s respiratory protein hemocyanin plays a central role in wood digestion and may offer a route toward biorefining of woody plant biomass.

Thermobia domestica belongs to an ancient group of insects and has a remarkable ability to digest crystalline cellulose without microbial assistance. By investigating the digestive proteome of Thermobia, we have identified over 20 members of an uncharacterized family of lytic polysaccharide monooxygenases (LPMOs). We show that this LPMO family spans across several clades of the Tree of Life, is of ancient origin, and was recruited by early arthropods with possible roles in remodeling endogenous chitin scaffolds during development and metamorphosis. Based on our in-depth characterization of Thermobia’s LPMOs, we propose that diversification of these enzymes toward cellulose digestion might have endowed ancestral insects with an effective biochemical apparatus for biomass degradation, allowing the early colonization of land during the Paleozoic Era. The vital role of LPMOs in modern agricultural pests and disease vectors offers new opportunities to help tackle global challenges in food security and the control of infectious diseases., LPMOs catalyze the oxidative breakdown of polysaccharides, thereby facilitating biomass degradation. By analyzing the digestive proteome of firebrats, the authors here identify a yet uncharacterized LPMO family and provide phylogenetic, structural and biochemical insights into its origin and functions.

Additional file 1. Additional tables, figures, and additional text.

Die Applikation von Salicylsäure (SA) und den synthetischen Analoga 2,6-Dichlorisonikotinsäure (DCINA) und Benzo(1,2,3)thiadiazol-7-carbothionsäure-S-methylester (BTH) führt in anfälligen Gerstensorten zu einem systemischen Schutz vor einer Infektion mit dem Erreger des Echten Gerstenmehltaus (Blumeria graminis f.sp. hordei, Bgh). Bei dieser chemisch Induzierten Resistenz (cIR) werden effektive Papillen und eine Hypersensitive Reaktion als Abwehrmechanismen nach Kontakt mit dem Pathogen verstärkt gebildet. In dieser Arbeit wurden verschiedene chemische und biotische Faktoren auf ihre resistenzinduzierende Wirkung überprüft. Es zeigte sich, dass die chemische Resistenzinduktion in der anfälligen Gerstensorte Manchuria, die kein bekanntes Resistenzgen besitzt, wirksam ist. Eine biotische Induktion der Resistenz gegenüber Bgh durch den avirulenten Echten Weizenmehltaupilz war dagegen in Manchuria nicht möglich. In einer molekularbiologischen Analyse der cIR wurden durch Suppressive Subtraktionshybridisierung Gene identifiziert, die nach Applikation chemischer Resistenzinduktoren differentiell exprimiert werden. Einige dieser Bci-Gene (barley chemically induced) waren in Gerste bereits als durch DCINA induzierbar bekannt, wie Lipoxygenase (Bci-1) und Thionin (Bci-2). Andere waren in Gerste bislang unbekannt und zeigen Homologie zu sauren Phosphatasen (Bci-3, Bci-6), zu Ca2+-bindenden single EF-hand-Proteinen (Bci-4), zu Serin-Proteinaseinhibitoren (Bci-7) und zu Apyrasen (Bci-9). Die Überprüfung der Geninduktion durch verschiedene abiotische und biotische Faktoren wie Phytohormone, Verwundung und Pathogene wurde zur Charakterisierung der Gene eingesetzt. Es stellte sich heraus, dass einige der Bci-Gene über verschiedene Signalwege der Pflanze induzierbar sind. In Übereinstimmung mit der im Vergleich zu BTH und DCINA schwächeren Resistenzinduktion gegenüber Bgh durch SA zeigte sich, dass auch die Induktion der Bci-Genexpression und insbesondere die von Bci-4 nach SA-Applikation schwächer war. Homologe von Bci-4 und anderen Bci-Genen waren in Weizen nachweisbar und dort ebenfalls chemisch induzierbar. Die Ergebnisse der Geninduktionsanalysen weisen darauf hin, dass sich besonders BCI-4 für den Einsatz als verlässlicher Marker der cIR in Getreide eignet. Ein BCI-4-Fusionsprotein mit dem grün fluoreszierenden Protein (GFP) als Reporter wurde in intakten Zwiebelepidermiszellen überexprimiert, wobei die subzelluläre Verteilung der GFP-Fluoreszenz auf eine Lokalisierung von BCI-4 im ER hindeutet. In einer Funktionsanalyse von BCI-4 konnte die Überexpression in Epidermiszellen anfälliger Gerstensorten die Resistenz gegenüber Bgh erhöhen. Damit stellt BCI-4 möglicherweise ein regulatorisches Element des Signaltransduktionsweges der cIR dar, das ausreicht, um Gerstenzellen in einen erhöhten Resistenzzustand zu versetzen. Application of salicylic acid (SA) and its synthetic mimicks 2,6-dichloroisonicotinic acid (DCINA) and benzo(1,2,3)thiadiazole-7-carbothioic acid S-methyl ester (BTH) protect susceptible barley plants systemically against infection with the powdery mildew fungus (Blumeria graminis f.sp. hordei, Bgh). Expression of chemically Induced Resistance (cIR) is associated with plant defense mechanisms that result in effective papilla formation and a hypersensitive response upon contact with the pathogen. In this study, different chemical and biotic factors were tested for their resistance inducing capacity. It was shown that chemical induction of resistance is efficient in Manchuria, though the cultivar does not bear any known resistance gene. In contrast, biotically Induced Resistance against Bgh with the non-host pathogen wheat powdery mildew fungus was not effective. Molecular analysis of cIR in barley using suppressive substraction hybridisation led to identification of genes that were differentially expressed upon application of chemical resistance inducers. Some of these Bci genes (barley chemically induced) coding for a lipoxygenase (Bci-1) and a thionin (Bci-2) were already known to be DCINA inducible, whereas others are newly described here. They show homology to genes of acid phosphatases (Bci-3 and Bci-6), Ca2+ binding single EF-hand proteins (Bci-4), serine proteinase inhibitors (Bci-7) and apyrases (Bci-9). Analyses of Bci gene expression profiles upon contact with various abiotic and biotic factors like phytohormones, wounding and pathogens were used to characterize these genes in more detail. Some of them were inducible through different signaling pathways of the plant. Compared with DCINA and BTH, SA was shown to be a weak inducer of resistance against Bgh. This is in accordance with the weak induction of gene expression especially regarding Bci-4. The results of gene induction analyses indicate that BCI-4 might be useful as marker for cIR in cereals. A BCI-4 fusion with green fluorescent protein (GFP) as reporter was overexpressed in onion epidermal cells. The subcellular distribution of GFP fluorescence seemed to be associated with the ER. In a functional gene analysis, resistance against Bgh was enhanced in susceptible barley by overexpressing Bci-4 in epidermal cells. Thus, BCI-4 might be a regulatory element of the signal transduction pathway leading to cIR in barley that is sufficient to mediate an enhanced resistance status of the plants.